Tuberculosis is still killing millions of people worldwide annually and the emergence of drug-resistant strains rekindles the global concern on public health threatened by TB. The growing prevalence of drug-resistant TB highlights the need for an effective vaccine. BCG, the only licensed TB vaccine, does not reliably protect adults from pulmonary TB. Numerous experimental TB vaccine candidates are not fundamentally superior to BCG in terms of vaccine efficacy in animal models. Conventional approaches to vaccine development, including antigen and adjuvant discovery, may not produce a breakthrough TB vaccine; new paradigms of vaccination are needed. The immune system has regulatory mechanisms to curtail excessive activation and limit tissue damage caused by infections and autoimmunity. Regulatory factors produced by dendritic cells (DC) include cytokines (e.g. IL-10), DC ligands for T cell inhibitory receptors (e.g. PDL1 and PDL2), and intracellular signaling regulators (e.g. SOCS1, SOCS2, and IRAK-M). These act to limit DC-mediated activation of effector T cells and to promote the differentiation of regulatory T cells. Vaccine adjuvants are needed to activate DC for T cell priming, but these adjuvants simultaneously induce the expression of suppressive genes. We hypothesize that the maximum immunostimulatory potential of vaccines is limited by counter-regulatory signals stimulated by the adjuvant. To test that hypothesis we propose the innovative concept of using siRNA to shape the immune response to vaccination. To apply siRNA as a vaccine component, efficient in vivo siRNA delivery is essential. We have developed a novel method for in vivo gene silencing using Glucan encapsulated siRNA Particles (GeRP). The versatility of GeRPs is such that other charged molecules, including protein antigens and adjuvant compounds can be co-packaged with siRNA inside the glucan shells using layer-by-layer synthesis. The resulting particles allow co-delivery of a complete vaccine formula to a DC via the receptor Dectin-1. The first step for proof of concept is to show that silencing one or more candidate suppressive genes (SOCS1, SOCS2, IRAK-M, IL-10, PDL1, and PDL2) by GeRP enhances T cell responses to a vaccine. Experimental approaches include DCs transfected with targeted siRNA or control siRNA and then tested for their ability to activate ovalbumin (OVA)-specific OT-II transgenic T cells in vitro and to sensitize animals with a Mycobacterium tuberculosis antigen (Ag85A) in vivo. The next step will be the incorporation of the best performing siRNA sequences into GeRPs engineered to combine Ag85A, adjuvants, and siRNA in one particle that will ensure each DC sees all components. GeRP vaccine constructs will be tested for their capability to confer a protective immunity in mice against aerosol M. tuberculosis challenge. The efficacy of this novel vaccine will be evaluated for TB prophylaxis, as a post-BCG booster, and as a therapeutic vaccine in combination with antimicrobial therapy. We seek R21 support for the exploratory phase of this TB vaccine project to produce data to support an RO1 proposal with the ultimate goal of developing a GeRP TB vaccine suitable for human clinical trials. In addition to its immediate translational goal, our approach may be applicable more broadly to vaccines against other human pathogens and GeRP vaccination may be validated as a platform to probe pathways of T cell priming and immune regulation using RNAi.